SIGNIFICANCE ‘Health has been proposed as the result of the organisms ability to cope with and adapt to stresses from our environment (1). In regard to the adaptive stress response triggered in cells by electrophiles and oxidants, the transcription factor ‘nuclear factor erythroid 2-related factor 2 (Nrf2) has become known as the key molecule, or even a “master switch” (2). This is highlighted by the fact that the Keap1-Nrf2 pathway orchestrates more than 600 cytoprotective genes, which regulate cellular detoxification, the elimination of ROS, xenobiotic metabolism, and drug transport (3).
CRITICAL ISSUE While in healthy cells this mechanism provides a strategy for the cell to “detox” by expressing these protective enzymes, cancer cells apply the exact same tactic to ensure their survival. This “dark side” of Nrf2 has often been neglected when discussing the effect of (dietary) antioxidants and their potential benefit for health (4), which is why until now many promising dietary phytochemicals have failed as chemopreventives in randomized controlled trials, while others exhibited even harmful effects (5) (6).
AIM To elucidate the question of the influence of anti- and prooxidant dietary phytochemicals as “friends or foes” on the signaling transduction in a human cellular cancer model thus became the focus of this thesis. This project to an extent deciphers the effect of selected dietary phytochemicals on the Nrf2 pathway and on endogenous antioxidant systems. Most importantly, the doctoral thesis at hand aims to define the bidirectional direct and indirect anti-/prooxidative properties of nutrigenomic activators of Nrf2 and thereby their potential to activate the thioredoxin detoxification system and heme-oxygenase-1.
METHODS The hepatocyte has been described as “a systemic hub”, because it engages in the bodily metabolic demand, iron homeostasis and, most prominently, detoxification processes, which are all redox-regulated (7). Therefore HepG2 cells, a well characterized and robust liver cancer cell line, were employed, a model which in this field of research is denoted “state-of-the-art”. The parameters investigated were cell viability with a resazurin-based assay, anti- and prooxidant effect with cell-based assays using a peroxyl-radical (AAPH) and a fluorescing indicator (DCF) as well as the reporter cell line HepG2-ARE-bla. Moreover, HepG2 cells were subfractioned into their major (and relevant) compartments cytoplasm, nuclei, mitochondria and as such Western blotted to analyze changes in Nrf2-target protein expression, selecting heme oxygenase-1 (HO-1), thioredoxin-1 (Trx-1) and thioredoxin reductase-1 (TrxR-1) as candidates. Furthermore, to follow up on endogenous ROS-production and the cells redox states, mitochondrial membrane potential changes were detected with the confocal microscope and a fluorescing dye (TMRM), as well as the multi-plate reader utilizing a different indicator stain (m-MPI). To obtain some in vivo insights also, a Kaplan Maier analysis was performed on two Nrf2 target genes (Trx-1 and TrxR-1) and their influence on survival probability.
RESULTS Having established that quercetin (QUE) acts predominantly as a direct antioxidant by scavenging ROS (the peroxyl-radical AAPH), sulforaphane (SFN) is proven as a lead substance in protecting the cells from oxidative stress via Nrf2-dependent modulation of the thioredoxin endogenous redox system, and at the same time as a weak prooxidant. Epigallocatchin-3-gallate (EGCG) employs both strategies, as the cell tries to re-establish homeostasis, which proves that these three substances make a highly interesting match. The influence of SFN, QUE, and EGCG in combination revealed novel and promising results on the IC50 of these liver cancer cells, which was lowered significantly (after 24 hrs: 76.36 M for SFN only; 180.5 M for EGCG only; compared to 52.54 M of SFN when paired with 50 M EGCG), and thus, EGCG is shown to aggravate the anti-tumorigenic effect of SFN. Moreover, SFN plus EGCG raised HO-1 levels significantly ( 2.81-fold) as well as TrxR-1 ( 1.85-fold) in reduced monomeric form. Another significant effect of EGCG is demonstrated in its capability to lower Trx-1 levels in HepG2 cells. As shown in the Kaplan Maier analysis, Trx-1 is a protein, which if overexpressed in cancer patients lowers their survival probability. While revealing synergistic effects of these three lead substances on Nrf2-target protein expression, one novel and striking finding is also that TrxR-1, a crucial part of the “redoxisome”, occurs in two sizes [kDa] depending on the treatment: a monomeric 55 kDa form, which polymerizes upon oxidative stress and appears clearly visible at a bigger molecular weight of 110-120 kDa. As demonstrated in the paper at hand, this phenomenon is counteracted by QUE, the prime direct antioxidant tested. Moreover, this thesis presents a dual approach to assessing mitochondrial membrane potential and shows the effects of SFN, QUE, and EGCG in qualitative and quantitative analyses, as single compounds and in combinations, which revealed synergistic, antagonistic, additive, and indifferent effects. Overall, this project challenges, first of all, the “antioxidant hypothesis”, according to which oxidative stress can be overcome by dietary intake of antioxidant phytochemicals, and reveals how these can work either directly as ROS scavengers or indirectly via the Nrf2 pathway exemplifying their bidirectional functionality. Secondly, this paper also examines the “oxidant hypothesis”, by exploring and employing prooxidative modes to lower the survival probability of cancer cells and thereby yielding significant findings.
FUTURE PROSEPCTS Naturally, more detailed concentration-time-organelle resolved studies as a follow-up to our study are advisable. Ideally, future research will assess both individual significant markers of cellular status at biochemical or phenotypical level and next generation omics sequencing. Some results of this project promise therapeutic successes, but more pre-clinical tests, in particular under physiologically true oxygen conditions (known as “physoxia”), are advisable.